Search Results (78)

Mitochondria serve as an essential component in the maintenance of cardiac function, and targeting them may represent a promising approach to handling heart failure (HF). HF in this review refers to various etiologies, including ischemic cardiomyopathy, dilated cardiomyopathy, and hypertrophic cardiomyopathy, unless otherwise specified. Mitochondrial dysfunction, a distinctive feature of HF, leads to a progressive decrease in bioenergetic reserves due to switching of energy production from oxidation of fatty acids in mitochondria to glycolytic pathways. The main problem in developing methods to improve mitochondrial function lies in the fact that protein preparations injected through the bloodstream cannot enter cells through the plasma membrane. Modern gene therapy involving the delivery of missing genes to cells using adeno-associated virus (AAV) vectors has the potential to improve the function of cardiomyocytes (CMCs). This type of therapy aims to target proteins that have been lost, damaged, or altered due to pathological conditions in the myocardium. This review summarizes pathophysiological mechanisms associated with mitochondrial dysfunction, which is mainly caused by increased oxidative stress and impaired mitochondrial biodynamics under HF progression. It also addresses possible ways to modulate these processes using gene therapy. Special attention is paid to modern characteristics of AAVs that can be used as vectors for the efficient delivery of desired genes to CMCs. Full article

Recombinant adeno-associated viral vectors (rAAVs) are the leading gene delivery vehicles in clinical development, yet efficient nuclear delivery remains a major barrier to effective transduction. This limitation is partly due to the incomplete understanding of rAAV’s complex subcellular trafficking dynamics. Here, we establish a longitudinal confocal live-cell imaging workflow that tracks rAAV2 from 4 to 12 h post-transduction, paired with an automated 3D analysis pipeline that quantifies spatiotemporal vector distribution, cytoplasmic trafficking, nuclear accumulation, and transgene expression at single-cell resolution. We use this platform to evaluate the effects of vector dose, cell cycle progression, and the behavior of empty particles. We identify previously undescribed trafficking features associated with high transgene expression. Higher rAAV2 doses enhanced cytoplasmic trafficking and nuclear delivery, while cell cycle progression facilitated both trafficking efficiency and transgene expression. We also characterize empty rAAV2 particles, revealing distinct trafficking patterns and markedly reduced nuclear accumulation compared to genome-containing vectors. By uncovering new bottlenecks in rAAV transduction, this platform provides mechanistic insights and potential strategies to improve AAV-based gene therapy. Its generalizable design further supports broad applicability to other non-enveloped viruses. Full article

Background: Adeno-associated viruses (AAVs) are widely used vectors for in vivo gene therapy, but their standard storage at −80 °C limits deployment in regions lacking ultracold infrastructure. Strategies enabling stable AAV storage at higher temperatures are needed to support global distribution. Methods: Nine AAV serotypes were lyophilized in simple sucrose-based buffers. Post-lyophilization vector integrity was assessed by measuring in vitro transduction efficiency using a luciferase reporter in cell-based assays. Stability of selected serotypes (AAV2.5 and AAV6) was further evaluated over 8 weeks under varying storage temperatures. Results: Lyophilization in sucrose preserved transduction activity for all tested serotypes (AAV1, AAV2, AAV2.5, AAV3, AAV4, AAV5, AAV6, AAV8, AAV9, and AAVrh10). Notably, AAV2, AAV2.5, and AAV6 exhibited 3- to 6-fold increases in transduction, an effect attributable to the sucrose excipient rather than the lyophilization process itself. Long-term stability of lyophilized vectors varied by serotype, temperature, and vial-seal integrity. AAV6 retained full activity for at least 8 weeks when stored at 4 °C or −20 °C. Conclusions: AAV vectors can be effectively lyophilized in simple sucrose solutions, enabling storage at standard −20 °C freezer temperatures while maintaining functional activity. Optimization of lyophilization buffers and excipients may further extend AAV stability at higher temperatures, improving feasibility for global gene therapy deployment. Full article

Adeno-associated viruses (AAVs) are a leading vector for gene therapy, yet their clinical utility is limited by the lack of robust quality control methods to distinguish between empty (AAV_empty), partially loaded (AAV_partial), and fully DNA loaded (AAV_full) capsids. Current analytical techniques provide partial insights but remain limited in sensitivity, throughput, or resolution. Here we present a multimodal plasmonic nanopore sensor that integrates optical trapping with electrical resistive-pulse sensing to characterize AAV9 capsids at the single-particle level in tens of μL sample volumes and fM range concentrations. As a model system, we employed AAV9 capsids not loaded with DNA, capsids loaded with a self-complementary 4.7 kbp DNA (AAV_scDNA), and ones loaded with single-stranded 4.7 kbp DNA (AAV_ssDNA). Ground-truth validation was performed with analytical ultracentrifugation (AUC). Nanosensor data were acquired concurrently for optical step changes (occurring at AAV trapping and un-trapping) both in transmittance and reflectance geometries, and electrical nanopore resistive pulse signatures, making for a total of five data dimensions. The acquired data was then filtered and clustered by Gaussian mixture models (GMMs), accompanied by spectral clustering stability analysis, to successfully separate between AAV species based on their DNA load status (AAV_empty, AAV_partial, AAV_full) and DNA load type (AAV_scDNA versus AAV_ssDNA). The motivation for quantifying the AAV_empty and AAV_partial population fractions is that they reduce treatment efficacy and increase immunogenicity. Likewise, the motivation to identify AAV_scDNA population fractions is that these have much higher transfection rates. Importantly, the results showed that the nanosensor could differentiate between AAV_scDNA and AAV_ssDNA despite their identical masses. In contrast, AUC could not differentiate between AAV_scDNA and AAV_ssDNA. An equimolar mixture of AAV_scDNA, AAV_ssDNA and AAV_empty was also measured with the sensor, and the results showed the expected population fractions, supporting the capacity of the method to differentiate AAV load status in heterogeneous solutions. In addition, less common optical and electrical signal signatures were identified in the acquired data, which were attributed to debris, rapid entry re-entry to the optical trap, or weak optical trap exits, representing critical artifacts to recognize for correct interpretation of the data. Together, these findings establish plasmonic nanopore sensing as a promising platform for quantifying AAV DNA loading status and genome type with the potential to extend ultra-sensitive single-particle characterization beyond the capabilities of existing methods. Full article

Adeno-associated viral vectors have proven to be a safe and effective gene therapy delivery system. Over the past decade, the approval of AAV gene therapies made a revolution in treatment of severe hereditary diseases, including spinal muscular atrophy, AADC deficiency, and others. Recombinant AAV-based therapeutics are currently intended for single administration. Safety concerns arise from immune responses to AAV and the resulting transgene, which can render subsequent injections ineffective. It remains unclear whether patients who have received an AAV-based gene therapy will need re-administration in the future. Furthermore, since many people have neutralizing antibodies or memory T cells against AAV from natural infections, it is crucial to overcome pre-existing immunity. This review considers existing modern approaches aimed to overcome both pre-existing natural immunity and immunity obtained after the administration of a gene therapy drug, which include various modifications of the viral drug (capsid modification, codon optimization), the use of empty capsid traps, and pharmacological support (immunosuppressive corticosteroids, inhibitors of various branches of the immune response, nanoparticles, IgG-degrading enzymes). The goal of this review is to illustrate the importance of this challenge and to highlight potential strategies for overcoming immunity to AAV-based gene therapies, contributing to the development of a successful therapeutic gene delivery platform. Full article

Bats host a wide range of viruses, including several high-profile pathogens of humans and other animals. The COVID-19 pandemic raised the level of concern regarding the risk of spillover of bat-borne viruses to humans and, conversely, human-borne viruses to bats. From August 2020 to July 2021, we conducted viral surveillance on 254 bats from 10 species across urban, periurban, and rural environments in New Mexico, USA. We used a pan-coronavirus RT-PCR to assay rectal swabs and performed metagenomic sequencing on a representative subset of 14 rectal swabs and colon samples. No coronaviruses were detected by either RT-PCR or metagenomic sequencing. However, four novel viruses were identified: an adenovirus (proposed name lacepfus virus, LCPV), an adeno-associated virus (AAV), an astrovirus (AstV), and a genomovirus (GV). LCPV, detected in a big brown bat (Eptesicus fuscus), is more closely related to canine adenoviruses than to other bat adenoviruses, suggesting historical transmission between bats and dogs. All virus-positive bats were either juvenile or adult individuals captured in urban environments; none exhibited obvious clinical signs of disease. Our findings suggest limited or no circulation of enzootic coronaviruses or SARS-CoV-2 in southwestern U.S. bat populations during the study period. The discovery of a genetically distinct adenovirus related to canine adenoviruses highlights the potential for cross-species viral transmission and underscores the value of continued virome surveillance in animals living with and near humans. Full article

Phenylketonuria (PKU) is an inherited disorder caused by mutations in the phenylalanine hydroxylase (PAH) gene that result in the amino acid phenylalanine (Phe) building up in the blood. Current therapies suggest low-Phe dietary management and (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH₄) therapy, which are limited in efficacy and require lifelong treatment. Recent advances in gene therapy, including gene editing and viral-mediated gene delivery, produce therapeutic effects. Advancements in gene editing technologies, notably adenine base editors (ABEs) and CRISPR-based systems, in conjunction with enhanced delivery methods such as lipid nanoparticles (LNPs) and recombinant viruses, have demonstrated substantial promise in preclinical studies. This review details the pathophysiology of PKU treatment, and progress in preclinical and clinical gene therapy strategies. Emphasis is on adenine base editing using LNPs, recombinant adeno-associated virus (rAAV)-mediated gene transfer, and the translational challenges associated with these technologies. We also discuss future directions for therapeutic reach and ensuring long-term safety and efficacy. Full article

Age-related macular degeneration (AMD) is a leading cause of irreversible vision loss in the elderly, characterized by progressive degeneration of the retinal pigment epithelium (RPE) and photoreceptors in the macula. Current treatment options primarily focus on slowing disease progression in neovascular AMD, while effective therapies for dry AMD remain limited. Optogenetics, a revolutionary technique utilizing light-sensitive proteins (opsins) to control the activity of genetically targeted cells, has emerged as a promising therapeutic strategy for restoring vision in retinal degenerative diseases. In retinal disease models, adeno-associated viruses (AAVs) serve as delivery vectors via intravitreal or subretinal injections. This review explores the principles of optogenetics, its application in preclinical AMD models, and the potential for clinical translation of this approach. We discuss the various optogenetic tools, delivery methods, and the challenges and future directions in harnessing this technology to combat AMD-related vision loss. Full article

Purpose. Genetic engineering of mesenchymal stromal cells (MSCs) using viral vectors has emerged as a promising approach to enhance the efficacy of anti-angiogenic gene therapies. Umbilical cord-derived MSCs are an attractive cell source due to their easy accessibility and potential for genetic modification. Adeno-associated viruses (AAVs) have been utilized in clinical settings to deliver therapeutic genes due to its characteristic of transient integration into the genome. In this study, we investigated the efficacy of using recombinant AAV-engineered umbilical cord-derived MSCs overexpressing anti-angiogenic factor, hsFlt-1 (MSCs.hsFlt1). Methods. The plasmid containing the hsFlt-1 gene was cloned into the AAV2 target backbone and validated using Sanger sequencing. The transduction process was studied to determine the optimal conditions, including the effect of MOI, media serum percentage, and attachment of MSCs, to achieve higher transduction efficiency. The functionality of MSCs.hsFtl1 was analyzed using qPCR, ELISA, and tube formation assays. Results. MSCs.hsFtl1 transduced at an MOI of 1 × 10⁶ demonstrated high transduction efficiency and exhibited robust gene and protein expression of hsFlt-1. The results revealed significant inhibition of growth in human umbilical vein endothelial cells (HUVECs) using a remarkably low dose of MSCs.hsFlt1 at 12.3 ng/mL. This observed anti-angiogenic effect was comparable to the clinically used Bevacizumab. Conclusions. The anti-angiogenic potential of MSCs.hsFlt1 effectively demonstrated in this study suggests their promising utility for targeted anti-angiogenic gene therapy approaches. Full article

Parvoviruses are small, single-stranded DNA viruses that have evolved sophisticated mechanisms to hijack host cell machinery for replication and persistence. One critical aspect of this interaction involves the manipulation of the host’s DNA Damage Response (DDR) pathways. While the viral genome is comparatively simple, parvoviruses have developed strategies that cause significant DNA damage, activate DDR pathways, and disrupt the host cell cycle. This review will explore the impact of parvovirus infections on host genome stability, focusing on key viral species such as Adeno-Associated Virus (AAV), Minute Virus of Mice (MVM), and Human Bocavirus (HBoV), and their interactions with DDR proteins. Since parvoviruses are used as oncolytic agents and gene therapy vectors, a better understanding of cellular DDR pathways will aid in engineering potent anti-cancer agents and gene therapies for chronic diseases. Full article

As the blood–brain barrier (BBB) prevents molecules from accessing the central nervous system (CNS), the traditional systemic delivery of chemical drugs limits the development of neurological drugs. However, in recent years, innovative therapeutic strategies have tried to bypass the restriction of traditional drug delivery methods. In vivo gene therapy refers to emerging biopharma vectors that carry the specific genes and target and infect specific tissues; these infected cells and tissues then undergo fundamental changes at the genetic level and produce therapeutic proteins or substances, thus providing therapeutic benefits. Clinical and preclinical trials mainly utilize adeno-associated viruses (AAVs), lentiviruses (LVs), and other viruses as gene vectors for disease investigation. Although LVs have a higher gene-carrying capacity, the vector of choice for many neurological diseases is the AAV vector due to its safety and long-term transgene expression in neurons. Here, we review the basic biology of AAVs and summarize some key issues in recombinant AAV (rAAV) engineering in gene therapy research; then, we summarize recent clinical trials using rAAV treatment for neurological diseases and provide translational perspectives and future challenges on target selection. Full article

Gene therapy offers promising potential as an efficacious and long-lasting therapeutic option for genetic conditions, by correcting defective mutations using engineered vectors to deliver genetic material to host cells. Among these vectors, adeno-associated viruses (AAVs) stand out for their efficiency, versatility, and safety, making them one of the leading platforms in gene therapy. The enormous potential of AAVs has been demonstrated through their use in over 225 clinical trials and the FDA’s approval of six AAV-based gene therapy products, positioning these vectors at the forefront of the field. This review highlights the evolution and current applications of AAVs in gene therapy, focusing on their clinical successes, ongoing developments, and the manufacturing processes required for the rapid commercial growth anticipated in the AAV therapy market. It also discusses the broader implications of these advancements for future therapeutic strategies targeting more complex and multi-systemic conditions and biological processes such as aging. Finally, we explore some of the major challenges currently confronting the field. Full article

In the dynamic field of gene therapy, recombinant adeno-associated viruses (rAAVs) have become leading viral vectors due to their safety, long-term expression, and wide-ranging cell and tissue tropism. With numerous FDA approvals and commercial products underscoring their potential, there is a critical need for efficient production processes to achieve high vector titers and quality. A major challenge in rAAV production is the efficient packaging of the genome into the viral capsid, with empty or partially filled capsids often representing over 90% of the produced material. To tackle this issue, we engineered the replication and packaging proteins of an AAV (Rep) to boost their functionality and improve vector titers. We subjected a complex Rep library derived from the AAV serotypes 1–13 to directed evolution in an AAV producer cell line. After each round of selection, single clones were analyzed, showing enrichment of specific hybrid Rep domains. Comparative analysis of these selected clones revealed considerable differences in their ability to package AAV2-based viral genomes, with hybrid Rep proteins achieving up to a 2.5-fold increase in packaging efficiency compared to their parental counterparts. These results suggest that optimizing rep gene variants through directed evolution is an effective strategy to enhance rAAV production efficiency. Full article

Adeno-associated viruses (AAVs) are non-pathogenic, replication-deficient viruses that have gained widespread attention for their application as gene therapy vectors. While these vectors offer high transduction efficiency and long-term gene expression, the host immune response poses a significant challenge to their clinical success. This review focuses on the obstacles to evaluating the humoral response to AAVs. We discuss the problems with the validation of in vitro tests and the possible approaches to overcome them. Using published data on neutralizing titers of AAV serotypes, we built the first antigenic maps of AAVs in order to visualize the antigenic relationships between varying serotypes. Full article

Most rare diseases (RDs) encompass a diverse group of inherited disorders that affect millions of people worldwide. A significant proportion of these diseases are driven by functional haploinsufficiency, which is caused by pathogenic genetic variants. Currently, most treatments for RDs are limited to symptom management, emphasizing the need for therapies that directly address genetic deficiencies. Recent advancements in gene therapy, particularly with adeno-associated viruses (AAVs) and lipid nanoparticle-encapsulated messenger RNA (mRNA), have introduced promising therapeutic approaches. AAV vectors offer durable gene expression, extensive tissue tropism, and a safety profile that makes them a leading choice for gene delivery; however, limitations remain, including packaging size and immune response. In contrast, mRNA therapeutics, formulated in LNPs, facilitate transient protein expression without the risk of genomic integration, supporting repeated dosing and pharmacokinetic control, though with less long-term expression than AAVs. This review analyzes the latest developments in AAV and mRNA technologies for rare monogenic disorders, focusing on preclinical and clinical outcomes, vector design, and delivery challenges. We also address key regulatory and immunological considerations impacting therapeutic success. Together, these advancements in AAV and mRNA technology underscore a new era in RD treatment, providing innovative tools to target the genetic root of these diseases and expanding therapeutic approaches for patients who currently face limited medical options. Full article